Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the sequence listing and the claims.
Neuropeptide Y (NPY) is a member of the pancreatic polypeptide family with widespread distribution throughout the mammalian nervous system. NPY and its relatives (peptide YY or PYY, and pancreatic polypeptide or PP) elicit a broad range of physiological effects through activation of at least five G protein-coupled receptor subtypes known as Y1, Y2, Y3, Y4 (or PP), and the "atypical Y1". The role of NPY as the most powerful stimulant of feeding behavior yet described is thought to occur primarily through activation of the hypothalamic "atypical Y1" receptor. This receptor is unique in that its classification was based solely on feeding behavior data, rather than radioligand binding data, unlike the Y1, Y2, Y3, and Y4 (or PP) receptors, each of which were described previously in both radioligand binding and functional assays. Applicants now report the use of a .sup.125 I-PYY-based expression cloning technique to isolate a rat hypothalamic cDNA encoding an "atypical Y1" receptor referred to herein as the Y5 subtype. Applicants also report the isolation and characterization of a Y5 homolog from human hippocampus. Protein sequence analysis reveals that the Y5 receptor belongs to the G protein-coupled receptor superfamily. Both the human and rat homolog display .ltoreq.42% identity in transmembrane domains with the previously cloned "Y-type" receptors. Rat brain localization studies using in situ hybridization techniques verified the existence of Y5 receptor mRNA in rat hypothalamus. Pharmacological evaluation revealed the following similarities between the Y5 and the "atypical Y1" receptor. 1) Peptides bound to the Y5 receptor with a rank order of potency identical to that described for the feeding response: NPY.gtoreq.NPY.sub.2-36 =PYY=[Leu.sup.31, Pro.sup.34 ]NPY&gt;&gt;NPY.sub.13-36. 2) The Y5 receptor was negatively coupled to cAMP accumulation, as had been proposed for the "atypical Y1" receptor. 3) Peptides activated the Y5 receptor with a rank order of potency identical to that described for the feeding response. 4) The reported feeding "modulator" [D-Trp.sup.32 ]NPY bound selectively to the Y5 receptor and subsequently activated the receptor. 5) Both the Y5 and the "atypical Y1" receptors were sensitive to deletions or modifications in the midregion of NPY and related peptide ligands. These data support the identity of the Y5 receptor as the previously described "atypical Y1", and furthermore indicate a role for the Y5 receptor as a potential target in the treatment of obesity, metabolism, and appetite disorders.
The peptide neurotransmitter neuropeptide Y (NPY) is a 36 amino acid member of the pancreatic polypeptide family with widespread distribution throughout the mammalian nervous system. NPY is considered to be the most powerful stimulant of feeding behavior yet described (Clark et al., 1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984). Direct injection into the hypothalamus of satiated rats, for example, can increase food intake up to 10-fold over a 4-hour period (Stanley et al., 1992). The role of NPY in normal and abnormal eating behavior, and the ability to interfere with NPY-dependent pathways as a means to appetite and weight control, are areas of great interest in pharmacological and pharmaceutical research (Sahu and Kalra, 1993; Dryden et al., 1994). Any credible means of studying or controlling NPY-dependent feeding behavior, however, must necessarily be highly specific as NPY can act through at least 5 pharmacologically defined receptor subtypes to elicit a wide variety of physiological functions (Dumont et al., 1992). It is therefore vital that knowledge of the molecular biology and structural diversity of the individual receptor subtypes be understood as part of a rational drug design approach to develop subtype selective compounds. A brief review of NPY receptor pharmacology is summarized below and also in Table 1.
TABLE 1: Pharmacologically defined receptors for NPY and related pancreatic polypeptides.
Rank orders of affinity for key peptides (NPY, PYY, PP, [Leu.sup.31, Pro.sup.34 ]NPY, NPY.sub.2-36, and NPY.sub.13-36) are based on previously reported binding and functional data (Schwartz et al., 1990; Wahlestedt et al., 1991; Dumont et al., 1992; Wahlestedt and Reis, 1993). Data for the Y2 receptor were disclosed in U.S. patent application Ser. No. 08/192,288 filed on Feb. 3, 1994, now U.S. Pat. No. 5,545,549, the foregoing contents of which are hereby incorporated by reference. Data for the Y4 receptor were disclosed in U.S. patent application Ser. No. 08/176,412 filed on Dec. 28 1993, now U.S. Pat. No. 5,516,653, the contents of which are hereby incorporated by reference. Missing peptides in the series reflect a lack of published information.
TABLE 1 ______________________________________ Recep- Affinity (pK.sub.i or pEC.sub.50) tor 11 to 10 10 to 9 9 to 8 8 to 7 7 to 6 &gt;6 ______________________________________ YI NPY NPY.sub.2-36 NPY.sub.13-36 PP PYY [Leu.sup.31, Pro.sup.34 ] NPY Y2 PYY NPY.sub.13-36 [Leu.sup.31, NPY Pro.sup.34 ] NPY.sub.2-36 NPY PP Y3 NPY [Pro.sup.34 ] NPY.sub.13-36 PP PYY Y4 PP PYY NPY NPY.sub.13-36 [Leu.sup.31, NPY.sub.2-36 Pro.sup.34 ] NPY atypical PYY NPY.sub.13-36 Y1 NPY (feed- NPY.sub.2-36 ing) [Leu.sup.31, Pro.sup.34 ] NPY ______________________________________
NPY Receptor Pharmacology
NPY receptor pharmacology has historically been based on structure/activity relationships within the pancreatic polypeptide family. The entire family includes the namesake pancreatic polypeptide (pp), synthesized primarily by endocrine cells in the pancreas; peptide YY (PYY), synthesized primarily by endocrine cells in the gut; and NPY, synthesized primarily in neurons (Michel, 1991; Dumont et al. , 1992; Wahlestedt and Reis, 1993). All pancreatic polypeptide family members share a compact structure involving a "PP-fold" and a conserved C-terminal hexapeptide ending in Tyr.sup.36 (or Y.sup.36 in the single letter code). The striking conservation of Y.sup.36 has prompted the reference to the pancreatic polypeptides' receptors as "Y-type" receptors (Wahlestedt et al., 1987), all of which are proposed to function as seven transmembrane-spanning G protein-coupled receptors (Dumont et al., 1992).
The Y1 receptor recognizes NPY.gtoreq.PYY&gt;&gt;PP (Grundemar et al., 1992). The receptor requires both the N- and the C-terminal regions of the peptides for optimal recognition. Exchange of Gln.sup.34 in NPY or PYY with the analogous residue from PP (Pro.sup.34), however, is well-tolerated. The Y1 receptor has been cloned from a variety of species including human, rat and mouse (Larhammar et al, 1992; Herzog et al, 1992; Eva et al, 1990; Eva et al, 1992). The Y2 receptor recognizes PYY.about.NPY&gt;&gt;PP and is relatively tolerant of N-terminal deletion (Grundemar et al., 1992). The receptor has a strict requirement for structure in the C-terminus (Arg.sup.33 -Gln.sup.34 -Arg.sup.35 -Tyr.sup.36 -NH.sub.2); exchange of Gln.sup.34 with Pro.sup.34, as in PP, is not well tolerated. The Y2 receptor has recently been cloned (disclosed in U.S. patent application Ser. No. 08/192,288, filed Feb. 3, 1994). The Y3 receptor is characterized by a strong preference for NPY over PYY and PP (Wahlestedt et al., 1991). [Pro.sup.34 ]NPY is reasonably well tolerated even though PP, which also contains Pro.sup.34, does not bind well to the Y3 receptor. The Y3 receptor (Y3) has not yet been cloned. The Y4 receptor (disclosed in U.S. patent application Ser. No. 08/176,412, filed Dec. 28, 1993, now U.S. Pat. No. 5,516,653) binds PP&gt;PYY&gt;NPY. Like the Y1, the Y4 requires both the N- and the C-terminal regions of the peptides for optimal recognition (U.S. Ser. No. 08/176,412, now U.S. Pat. No. 5,516,653). The "atypical Y1" or "feeding" receptor was defined exclusively by injection of several pancreatic polypeptide analogs into the paraventricular nucleus of the rat hypothalamus which stimulated feeding behavior with the following rank order: NPY.sub.2-36 .gtoreq.NPY.about.PYY.about.[Leu.sup.31,Pro.sup.34 ]NPY&gt;NPY.sub.13-36 (Kalra et al., 1991; Stanley et al., 1992). The profile is similar to that of a Y1-like receptor except for the anomalous ability of NPY.sub.2-36 to stimulate food intake with potency equivalent or better than that of NPY. A subsequent report in J. Med. Chem. by Balasubramaniam et al. (1994) showed that feeding can be regulated by [D-Trp.sup.32 ]NPY. While this peptide was presented as an NPY antagonist, the published data at least in part support a stimulatory effect of [D-Trp.sup.32 ]NPY on feeding. [D-Trp.sup.32 ]NPY thereby represents another diagnostic tool for receptor identification. In contrast to other NPY receptor subtypes, the "feeding" receptor has never been characterized for peptide binding affinity in radioligand binding assays and the fact that a single receptor could be responsible for the feeding response has been impossible to validate in the absence of an isolated receptor protein; the possibility exists, for example, that the feeding response could be a composite profile of Y1 and Y2 subtypes.
Applicants now report the isolation by expression cloning of a novel Y-type receptor from a rat hypothalamic cDNA library, along with its pharmacological characterization, in situ localization, and human homologues. The data provided link this newly-cloned receptor subtype, from now on referred to as the Y5 subtype, to the "atypical Y1" feeding response. This discovery therefore provides a novel approach, through the use of heterologous expression systems, to develop a subtype selective antagonist for obesity and other indications.
Applicants further report the isolation of a canine Y5 receptor.
In addition, applicants report the discovery of chemical compounds which bind selectively to the Y5 receptor of the present invention and which act as antagonists of the Y5 receptor.
The treatment of disorders or diseases associated with the inhibition of the Y5 receptor subtype, especially diseases caused by eating disorders like obesity, bulimia nervosa, diabetes, dislipidimia, may be effected by administration of compounds which bind selectively to the Y5 receptor and inhibit the activation of the Y5 receptor. Furthermore, any disease states in which the Y5 receptor subtype is involved, for example, memory loss, epileptic seizures, migraine, sleep disturbance, and pain, may also be treated using compounds which bind selectively to the Y5 receptor.